Abstract

Apoptosis, or programmed cell death, is an important mechanism by which
cells are eliminated during immune regulation and embryonic
development. Aberrations in the signaling pathways leading to apoptosis
may result in cancer, autoimmune diseases, or inflammatory disorders.
In view of this, an understanding of the signaling capabilities of
apoptosis-inducing or death receptors is essential to understanding
their roles in biology and disease. We used cDNA microarrays to examine
the downstream transcriptional effects of two members of the tumor
necrosis factor (TNF) family of death receptor ligands. We compared the
transcriptional responses of a model colon cancer cell line, HT29, to
TNF-α and anti-Fas activating antibody. Both ligands induced a subset
of genes characteristic of activation of the transcription
factor nuclear factor-κB (NF-κB). Follow-up analyses demonstrated
that, although TNF-α activated NF-κB through IκB-α degradation,α
-Fas treatment led to NF-κB activation through a mechanism
distinct from IκB-α degradation.

Introduction

TNF
3
-α and Fas ligand are two members of a growing superfamily of ligands
that bind to their cognate receptors, TNF-R and Fas (CD95), on the cell
surface and transmit downstream signals. Both ligands, upon binding to
their cognate membrane receptor, cause recruitment of adapter molecules
that in turn activate caspases and other proapoptotic signals,
eventually leading to cell death
(1)
. TNF-α is also able
to elicit a pleiotropic response in most cell types through
transcriptional activation of a wide variety of genes
(2)
.
Many of these target genes are implicated in immune and inflammatory
responses. One of the downstream targets of TNF receptors is the
transcription factor NF-κB. NF-κB is sequestered in the cytoplasm
by IκB, and upon stimulation by TNF-α, IκB is phosphorylated,
ubiquitinated, and targeted to the proteosome for degradation. The free
NF-κB dimer, usually consisting of p65 and p50 subunits, then
translocates to the nucleus and binds to consensus elements within the
promoters of a variety of genes
(3)
. Among the genes
activated by NF-κB are genes thought to oppose the proapoptotic
effects of TNF. IAPs, for example, are rapidly induced in response to
TNF-α and are thought to delay apoptosis through direct inhibition of
caspase activity
(4)
. Previous reports have shown that Fas
activation can induce secretion of the inflammatory cytokine IL-8, a
known NF-κB-responsive gene
(5)
. Furthermore, previous
studies have reported that Fas ligation by an agonistic antibody
activates NF-κB DNA binding in certain cell types
(6)
and that Fas ligation can activate the c-Jun NH2
kinase pathway in human colonic epithelial cells
(7)
.
Little is known, however, about transcriptional responses to activation
of Fas. It is widely believed that the Fas receptor is a “pure
killer” in that its activation of caspases and the resulting
apoptosis require no transcriptional component. The ability of TNF-α
to elicit dual responses of proliferation and cell death, depending on
conditions, raises the question as to whether other members of the TNF
family, like Fas ligand, are able to do the same.

In light of the evidence that there appear to be cell types and/or
conditions under which Fas ligation is able to activate transcriptional
pathways similar to TNF-α, we decided to examine the scope of this
effect by comparing the downstream transcriptional profiles elicited by
TNF-α and anti-Fas. We used cDNA microarrays, containing 4555
minimally redundant genes, to compare the expression profiles
downstream of the TNF and Fas receptors in the colon adenocarcinoma
cell line, HT29. Both agents were able to induce a largely overlapping
set of genes, including a subset of known NF-κB targets like
cIAP-2.

Microarray Experiments.

Microarray slides were produced as described previously
(8)
. HT29 cells were treated with 10 ng/ml IFN-γ for
16 h and then with 100 ng/ml TNF-α or 100 ng/ml α-Fas antibody
for 4 h. IFN-γ was maintained in the medium during the treatment
period with each ligand. Total RNA was isolated using Trizol reagent
(Life Technologies, Inc.), and poly(A) RNA was selected with Oligotex
(Qiagen) according to the manufacturers’ protocols. Labeling and
hybridization were performed as described previously
(8)
.
Briefly, first-strand cDNA probes were generated by incorporation of
Cy3-dCTP or Cy5-dCTP (Amersham Pharmacia) during reverse transcription
of 1 μg of purified mRNA. The resulting cDNA probes were purified by
vacuum filtration, denatured at 94°C, and hybridized to an arrayed
slide overnight at 42°C. Slides were washed in 1× SSC/0.2% SDS for
10 min and then in 0.1× SSC/0.2% SDS for 20 min. Slides were rinsed
and dried, and fluorescence was captured using the Avalanche dual laser
confocal scanner (Molecular Dynamics). Fluorescent intensities were
quantified using Arrayvision 4.0 (Imaging Research).

Microarray Data Analyses.

The software program GeneSpring (version 3.2.2; Silicon Genetics) was
used to analyze the array data as follows. The raw fluorescence units
of genes represented more than once on each slide were averaged. The
ratio was then taken of the signal (IFN-γ plus TNF-α orα
-Fas):control (IFN-γ only). To normalize for variation among
slides, the ratios (death ligand-treated:control) from each replicate
slide were normalized to 1.0 (ratio of gene A in experiment X:median of
all ratios measured in experiment X). A Student’s t test
was performed to calculate whether the mean relative intensity for a
gene was statistically different from 1.0. Control-only comparisons
were used to establish significance values for determining induced or
repressed genes. For up-regulated genes, we used a significance cutoff
of 1.6 (average log ratio >0.204). For down-regulated genes, the
significance value was set at 0.45 (average log ratio less than−
0.347). The genes exceeding the set significance values, which also
had P < 0.085, are presented.

Northern Analyses.

HT29 cells were treated as above for microarray experiments. Total RNA
was harvested at appropriate times after treatment, and 7 μg were run
on a denaturing formaldehyde agarose gel. RNA was transferred to nylon
membrane (Amersham Pharmacia) and cross-linked. Blots were
prehybridized at 65°C for 1 h in Rapid-Hyb buffer (Amersham
Pharmacia) and then hybridized with random-primed,[α
-32P]dCTP-labeled probes for 4–16 h at
65°C. Unbound probe was removed by washing twice in 2× SSC/0.1% SDS
at room temperature and then twice in 0.5× SSC/0.1% SDS at 65°C.
Blots were exposed to a phosphor screen and scanned using a
Phosphorimager (Molecular Dynamics).

IκB Western Blots.

Whole-cell extracts were prepared from treated HT29 cells as follows.
Cells were washed twice in cold PBS, incubated on ice for 10 min in
lysis buffer [25 mm Tris-HCl (pH 7.4), 150 mm
NaCl, 1 mm CaCl2, 1% Triton X-100, 1
mm PMSF, 10 μg/ml leupeptin, and 10 μg/ml aprotinin],
scraped from plate into tube, and spun at 14,000 rpm for 10 min at
4°C. Supernatants were removed and assayed for protein concentration
using Bio-Rad protein assay kit. Protein extracts were analyzed using
SDS-PAGE on 10% Tris-glycine gels (Novex) and transferred to
polyvinylidene difluoride membranes (Gelman Sciences). Blots were
incubated 1 h at room temperature in blocking buffer (5% powdered
milk, PBS, and 0.1% Tween 20) and then with a 1:1000 dilution of
either a polyclonal antibody to IκB-α or a phosphospecific antibody
to serine 32 of IκB-α (New England Biolabs) in primary antibody
buffer (5% BSA, PBS, and 0.1% Tween 20) overnight. Blots were washed
three times in PBS/0.1% Tween 20 and then probed with a secondary
antibody conjugated to horseradish peroxidase (Life Technologies, Inc.)
for 30 min. After washing three times in PBS/0.1% Tween 20, protein
was detected using a chemiluminescent substrate (DuPont NEN Life
Sciences) according to manufacturer’s protocol.

Results and Discussion

IFN-γ in combination with TNF-α or α-Fas antibody act
synergistically to induce apoptosis in a variety of cell types
(5)
. To establish a concentration curve from which to
select treatment conditions for microarray analyses, we preincubated
HT29 colon adenocarcinoma cells with 10 ng/ml IFN-γ for 16 h and
then treated these cells with increasing concentrations of TNF-α or
the agonistic α-Fas CH-11 antibody. We then assayed for cell death by
measuring uptake of YO-PRO-1 dye. As shown in Fig. 1
⇓
, when cells were pretreated with IFN-γ, both TNF-α and α-Fas
induced killing in a saturable, concentration-dependent manner. Because
cells without IFN-γ treatment were relatively resistant to killing
mediated by either receptor, our subsequent experiments included
pretreatment of the cells with IFN-γ.

Concentration-dependent cell death in HT29 cells. HT29
cells were treated with vehicle (▪) or with 10 ng/ml IFN-γ (▴)
for 16 h and then with increasing concentrations of soluble
TNF-α (A) or α-Fas (CH11 antibody; B)
for 24 h. Cell death was measured by YO-PRO-1 fluorescence.
Bars, SD.

To examine the transcriptional pathways activated downstream of the TNF
and Fas receptors, we used cDNA microarrays
(9)
to monitor
transcriptional changes in HT29 cells upon treatment with TNF-α orα
-Fas antibody. HT29 cells, preincubated with IFN-γ for 16 h,
were treated with vehicle or a saturating concentration of either
ligand for 4 h. Each culture, vehicle or ligand-treated, contained
an equal concentration of IFN-γ during the treatment period. After
incubation, mRNA was harvested and used to generate labeled cDNA probes
(IFN-γ alone or IFN-γ plus either TNF-α or α-Fas). The probes
were simultaneously hybridized to a microarray slide containing 4555
cDNAs in duplicate (Fig. 2A)
⇓
. In addition, to assess the technical capabilities of our
arrays, we generated probes labeled with each dye from untreated,
control HT29 cells and cohybridized them in parallel with our
experimental samples. Slides were analyzed by quantifying fluorescence
intensities and plotting the fluorescence of control (IFN-γ only)
versus treatment (IFN-γ plus TNF-α or α-Fas; Fig. 2B⇓
). We then calculated the log of the relative intensity
ratios (IFN-γ plus TNF-α or α-Fas:IFN-γ only) and averaged the
four replicates. Control-only comparisons were used to establish
significance values for determining induced or repressed genes. On the
basis of this, we selected genes displaying expression ratios of >1.6
(log value >0.204) or <0.45 (log value less than −0.347), which also
had Ps of <0.085. These genes were sequence verified and
are presented in Table 1
⇓
.

Microarray analyses and temporal induction of two
representative genes. A, mRNAs from control HT29 cells
(upper panel), IFN-γ-primed HT29 cells treated 100
ng/ml TNF-α (middle panel), or IFN-γ primed HT29
cells treated with α-Fas antibody (lower panel) were
reverse-transcribed in the presence of Cy5-dCTP (red).
Corresponding samples from control (upper panel) or
IFN-γ-primed cells (middle and lower
panels) were reverse-transcribed in the presence of Cy3-dCTP
(green) for the respective comparisons. Resulting cDNA
probes were cohybridized to a microarray containing 4555 target genes
in duplicate. A representative slide section of 384 genes is shown for
each treatment. B, changes in transcript levels were
quantified by measuring fluorescent signals and plotting control (IFN
only) versus treated (IFN plus TNF-α or α-Fas).
Red and green indicate overexpressed and
underexpressed, respectively. Left panels, signal
intensities for all 4555 array elements. Right panels,
only those genes meeting the criteria for induction (up 1.6-fold with
P < 0.085). Blue lines,
2-fold induction and repression as compared with the normalized median.
C, RNA from HT29 cells treated with or without IFN-γ
was harvested at various times after stimulation with TNF-α orα
-Fas as above and analyzed by Northern blot using PCR-amplified
probes. Transcripts running at 6.5 and 1.8 kb for cIAP-2
and IL-8, respectively, are shown along with
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a
loading control.

Striking in our analysis was that TNF-α treatment resulted in an
asymmetric distribution of induced versus suppressed genes.
Treatment of HT29 cells with TNF-α caused the induction of 29 genes
that exceeded our cutoff of 1.6 (log ≥0.204). In contrast, no genes
exceeded the suppression levels established in our control only
comparison. This indicates that the early transcriptional program
elicited by TNF is aimed primarily at induction of target genes. It is
possible that this distribution would change over time to generate a
more symmetrical distribution; however, these data demonstrate the
potential for asymmetry in the activation of transcriptional programs
by specific ligands.

We categorized the up-regulated genes into three main groups:

(a) Fifteen of the 29 genes were known to be TNF-α or
NF-κB regulated, including the cytokines IL-8 and
GRO-2 (GRO-β) and the antiapoptosis gene
apoptosis inhibitor 2 (cIAP-2; Ref.
10
). These genes served as positive internal controls and
confirmed the validity of our microarray approach.

(b) As a second category, 10 of the 30 genes regulated
by TNF-α were genes of known function that lacked previous
description, implicating them downstream of the TNF receptor.
Interestingly, genes in this category included genes that have been
reported to be antiapoptotic. For example, one of the genes,
14-3-3η (tyrosine 3-monooxygenase/tryptophan
5-monooxygenase activation protein), belongs to a family of
proteins that has been shown to inhibit cell death by binding to the
apoptosis-promoting proteins BAD and forkhead transcription factor
(FKHRL1; Ref.
11
).

(c) Finally, we observed four TNF-α-regulated genes of
unknown function that corresponded only to ESTs.

Several of the TNF-α-induced genes, including small inducible
cytokine member 10, Humig (monokine induced by IFN-γ), and
guanylate binding protein 2 are known targets of the IFN
response pathway
(8)
. It was possible, therefore, that the
induction of these genes by TNF-α was occurring through increased
cell sensitization to IFN-γ. We believe this unlikely in that many
other known IFN response genes were absent for the TNF-α-inducible
list
(8)
. To address this directly, we performed array
experiments wherein IFN-containing medium was removed and replaced with
medium containing the death ligands only. Under these conditions, we
saw the same TNF-α induction pattern, including those genes that are
also targets of IFN. Further investigations will be required to fully
characterize the integration of signaling pathways that emanate from
the TNF receptors and how these pathways couple with specific
transcription factor complexes.

Treatment of HT29 cells with α-Fas elicited a gene expression profile
that almost directly overlapped with TNF-α. Table 1
⇓
shows the genes
regulated by TNF-α and the response of these genes to α-Fas
antibody. In this analysis, six genes were induced by Fas ligation to a
level that exceeded the log expression ratio of 0.204. Five of the six
genes were also induced by TNF-α, suggesting a common signaling
pathway. There was only one gene (coagulation factor XIIIa)
that was highly induced by α-Fas that failed to meet the criteria for
induction by TNF-α. Our data indicate a general activation of NF-κB
response genes by α-Fas and extend the previous results showing that
Fas ligation stimulates IL-8 production
(5)
.

We saw many more genes that were repressed, relative to the control
comparison, in cells responding to α-Fas (54 genes) than were evident
in the TNF-α-treated cells (0 genes). Because both TNF-α andα
-Fas induce cell death in HT29 cells, we believe that many of these
repressed changes could be attributable to the cells’ metabolic state,
not attributable directly to repression. For example, basal levels of
transcription or message stability could contribute to a nonspecific
loss of certain mRNAs. Because we cannot distinguish between specific
and nonspecific message loss, we have elected to present only the
up-regulated targets of TNF-α andα
-Fas.
4

We then selected two genes, IL-8 and cIAP-2, with
roles in inflammation and cell death regulation for further analysis.
In addition, IL-8 and cIAP-2 were highly and
moderately induced, respectively, by both ligands. We performed
Northern blot analyses to monitor the temporal induction of each gene
and to confirm the induction seen by microarray analysis (Fig. 2C)
⇓
. HT29 cells pretreated with IFN-γ were treated with
saturating concentrations of TNF-α and α-Fas, and RNA was harvested
at various times after stimulation. TNF-α induced cIAP-2
and IL-8 rapidly, with maximal expression of both
transcripts occurring at 2 h. The kinetics of induction, however,
differed for the two transcripts, with cIAP-2 message
returning to basal level by 8 h and IL-8 remaining
elevated. The induction of both genes by TNF-α was only slightly
enhanced by pretreatment with IFN-γ. In contrast, α-Fas antibody
caused an induction of both genes that was temporally delayed in
comparison to TNF-α (Fig. 2C)
⇓
. In the case of Fas
ligation, maximal expression of both genes occurred at 6 h and was
sustained for at least 8 h. Unlike TNF-α, pretreatment with
IFN-γ was required for the induction of cIAP-2 and greatly
enhanced the induction of IL-8 by α-Fas.

The numerous NF-κB-regulated genes induced in our microarray
experiments led us to investigate whether α-Fas activated NF-κB DNA
binding in HT29 cells. To determine whether NF-κB DNA binding
activity was present in cells treated with α-Fas, we performed
electrophoretic mobility shift assays on nuclear extracts from HT29
cells (Fig. 3A)
⇓
. Binding to a NF-κB consensus oligonucleotide was
evident in both TNF-α- and α-Fas-treated extracts. Antibody
supershift experiments revealed that both p65 and p50 subunits of
NF-κB bound to the DNA in response to either TNF-α or α-Fas (Fig. 3A)
⇓
. Consistent with the transcriptional induction of
cIAP-2 and IL-8 (Fig. 2C)
⇓
, activation
of NF-κB by TNF-α was only slightly augmented by pretreatment of
the cells with IFN-γ. Induction of NF-κB by α-Fas, however, was
greatly enhanced by IFN-γ. The binding induced by α-Fas, however,
was weaker than that elicited by TNF-α, an observation that was
consistent with the weaker induction of response genes by α-Fas as
seen in the microarrays (Table 1)
⇓
.

Activation of NF-κB. A, nuclear protein
extracts (6 μg) from HT29 cells, treated with or without 10 ng/ml
IFN-γ plus 100 ng/ml TNF-α or α-Fas, were mixed with a
32P-labeled oligonucleotide containing a consensus NF-κB
binding site. Treatments are indicated above each lane.
For antibody (Ab) supershift experiments,
IFN-γ-treated extracts were incubated with 2 μg of antibody for 15
min prior to addition of labeled oligo. Shifted complexes are indicated
by arrows.n.s., nonspecific band.
B, HT29 cells were treated with 10 ng/ml IFN-γ for
16 h and then with 100 ng/ml TNF-α or α-Fas. Whole-cell
protein extracts prepared at various times after stimulation were
separated by SDS-PAGE, transferred to a polyvinylidene difluoride
membrane, and probed with a polyclonal antibody to IκB-α.

We next examined the mechanisms through which Fas ligation leads to
NF-κB activation. It was possible that α-Fas binds other cell
surface receptors, distinct from Fas, which cause NF-κB activation.
This was of particular concern, given the recent findings that several
agonistic Fas antibodies show cross-reactivity with other cellular
proteins
(12)
. To exclude this possibility, we pretreated
HT29 cells with a Fas-specific neutralizing antibody (ZB4; Upstate
Biotechnology, Inc.) that recognizes an antigen distinct from the CH-11
activating antibody. The cells were treated with 500 ng/ml ZB4 for
1 h prior to stimulation with 100 ng/ml CH-11 for 4 h. RNA
was harvested and analyzed by Northern blot. In these experiments,
neutralizing antibody protected the cells from Fas-mediated apoptosis
and inhibited Fas-mediated induction of IL-8 message by at least 70%.
This indicated that the CH-11 antibody elicited death and
transcriptional responses directly through Fas, not through nonspecific
interactions.

Another possible mechanism for indirect activation of NF-κB after Fas
ligation includes the processing of inflammatory cytokines by
activation of caspases. For example, caspases can process pro-IL-1 and
pro-IL-18 in response to Fas ligation and elicit an inflammatory
response
(13, 14)
. To address this possibility, we tested
whether inhibition of caspases prevented induction of NF-κB response
genes. We incubated HT29 cells with 10 μm
Z-Val-Ala-Asp-fluoromethyl ketone, a general inhibitor of caspases, for
1 h prior to stimulation with α-Fas for 4 h and then
measured the induction of cIAP-2 and IL-8 by
Northern analysis. Although the Z-Val-Ala-Asp-fluoromethyl ketone
blocked cell death, it failed to prevent induction of cIAP-2
or IL-8 (data not shown). This eliminated caspases as
generators of NF-κB activators and supported that Fas ligation was
directly causing gene transcription in HT29 cells.

The most well-described method of NF-κB activation occurs
through phosphorylation and subsequent degradation of IκB-α. We,
therefore, analyzed the levels of IκB-α at various times after
stimulation. Whole-cell lysates were prepared and analyzed by probing a
Western blot with a polyclonal antibody to IκB-α. As expected,
TNF-α caused a rapid degradation of IκB-α beginning at 5 min,
with the protein being completely degraded by 15 min (Fig. 3B)
⇓
. Surprisingly, α-Fas failed to cause any visible
IκB-α degradation up to 20 min after stimulation. Phosphorylation
of IκB-α by TNF-α was evident at 5 min, whereas there was no
detectable phosphorylation by α-Fas for up to 20 min (data not
shown). We then examined up to 4 h after stimulation by α-Fas
and did not observe degradation of IκB-α (data not shown). Although
lack of degradation was also true of IκB-β (data not shown), there
are several additional isoforms of IκB, including ε, γ, Bcl3,
p105, and p100
(10)
through which α-Fas may activate
NF-κB. The mechanism, however, appears distinct from that used by
TNF-α.

Our data show that microarrays are capable of establishing gene
expression profiles for members of the TNF family of death receptors.
From such signature patterns, we were able to determine that ligation
of the Fas receptor activates a NF-κB pathway, resulting in the
transcriptional induction of genes thought to be part of inflammatory
or survival pathways. Many human tumors overexpress Fas ligand
(15, 16)
. This has led to speculation that increased
levels of Fas ligand on the cell surface cause apoptosis of
infiltrating lymphocytes, allowing the tumor cells to escape immune
surveillance
(15,
16,
17)
. In contrast, it is thought that
NF-κB activation can elicit an antiapoptotic pathway by inducing the
transcription of “survival” genes
(18)
. Among these
are apoptosis inhibitor 2 (cIAP-2) and the
cytoprotective gene, manganese superoxide dismutase(SOD-2). We saw that each of these genes was
up-regulated by either TNF-α or α-Fas under the same conditions
that caused the cells to undergo apoptosis. Regulation of these genes
by α-Fas has not been reported and contradicts the widespread notion
that Fas signaling leads only to cell death. Fas-mediated activation of
NF-κB genes, as demonstrated here, supports a
role for Fas in inflammation. It is unclear, however, whether induction
of genes like cIAP-2 alone is sufficient to block
Fas-mediated apoptosis. Remacle-Bonnet et al.(19)
demonstrated recently the inability of insulin-like
growth factor I to protect HT29-D4 cells from Fas-mediated killing
unless the cells were simultaneously exposed to TNF-α. This suggests
that activation of pathways, like the mitogen-activated protein
kinase/extracellular signal-regulated kinase cascade may be required as
supplements to IAP-2 induction in blocking death receptor mediated
apoptosis. Induction of cIAP-2 by α-Fas, however, is in
agreement with studies demonstrating that transplanted Fas
ligand-bearing tumors elicit an inflammatory response rather than
conferring immune privilege
(20)
. Finally, our data
provide new insights that activation of Fas in some cancer cells can
elicit a response similar to the inflammatory response seen with
TNF-α. This suggests the possibility that overexpression of Fas
ligand in tumors may induce a chronic inflammatory response, which
could promote tumor development.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.